Prothrombinase complex assembly. Contributions of protein-protein and protein-membrane interactions toward complex formation.
Equilibrium binding studies of prothrombinase complex formation were undertaken using phospholipid vesicles composed of phosphatidylcholine and phosphatidylserine (PCPS), factor Va, and factor Xa modified with dansyl glutamylglycinylarginyl chloromethyl ketone (DEGR.Xa). The interaction between the Va.PCPS and DEGR.Xa.PCPS binary complexes was experimentally isolated using saturating concentrations of PCPS. Fluorescence titrations indicated that the membrane-bound proteins interact tightly (Kd approximately 10(-9) M) with a stoichiometry of 1 mol of Va bound/mol of DEGR.Xa at saturation. Complex formation was also investigated by kinetic studies of prothrombin activation using unmodified factor Xa. The kinetic studies yielded a Kd approximately 10(-9) M, which was independent of the concentration of prothrombin in the range of 0.5-5.0 microM. Fluorescence studies of complex assembly at limiting PCPS concentrations provided evidence for an altered DEGR.Xa-PCPS interaction when the enzyme was assembled into the complex. The data suggest that although both proteins are associated with PCPS when complexed with each other, the presence of factor Va on the membrane surface increases the affinity for the Xa-PCPS interaction by an estimated 100-fold. Prothrombinase complex assembly therefore proceeds independently of the availability of substrate and is stabilized by protein-protein and protein-phospholipid interactions. Linkage between the two protein-membrane combination events leads to the further stabilization of the complex on the vesicle surface.
Krishnaswamy
S
S
Department of Biochemistry, University of Vermont, Burlington 05405.
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003619:Dansyl Compounds / Q000494:pharmacology; D005165:Factor V / Q000378:metabolism*; D015943:Factor Va / Q000302:isolation & purification / Q000378:metabolism; D005170:Factor X / Q000378:metabolism*; D015951:Factor Xa / Q000378:metabolism; D007700:Kinetics; D008081:Liposomes; D008433:Mathematics; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D013050:Spectrometry, Fluorescence
D003619:Dansyl Compounds; D008081:Liposomes; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; C022475:prothrombinase complex; D015943:Factor Va; D005165:Factor V; D005170:Factor X; D015951:Factor Xa